PROJECT SUMMARY Approximately 400,000 people in the United States are living with multiple sclerosis (MS). MS is a neuroinflammatory disease that affects a disproportionate amount of young people between the ages of 20 and 40 years of age. Immune infiltration across the blood-brain barrier (BBB) into the brain parenchyma is one of the initial processes in the disease progression of multiple sclerosis and is poorly understood. We hypothesize that gaining a better understanding the mechanism of immune infiltration across the blood brain barrier and the contribution of various leukocyte subsets may lead to the discovery of new therapeutics for minimizing immune infiltration for multiple sclerosis and other inflammatory diseases of the brain. We propose to create, characterize, and validate our brain post-capillary venule platform and to use this platform to visualize and quantify the various steps in the mechanism of immune infiltration, as well as to investigate potential therapeutics for multiple sclerosis. This will be accomplished via the following Specific Aims. In Aim 1, a brain post-capillary venule platform will be designed that incorporates the physiological characteristics of brain post- capillary venules based on in vivo studies. These vessels will be characterized and analyzed using a toolbox of parameters to establish baseline conditions. In Aim 2, the mechanisms involved in leukocyte infiltration across the blood-brain barrier will be investigated. Using live-cell time-lapse microscopy, the multi-step process of leukocyte infiltration (including rolling, adhesion, diapedesis, cell fate etc.) of various leukocyte subsets will be visualized and quantified to assess the differential leukocyte infiltration. In addition, this platform will also be used to investigate potential therapeutics for multiple sclerosis, including testing minocycline, a well-known antibiotic with anti-inflammatory effects, and receptor blockers. Furthermore, this in vitro brain post-capillary venule platform has the potential to be modified and adapted to study many other central nervous system disorders that involve the blood-brain barrier.